Radiolabeling of catalytic subunits of pi 3-kinases with 17β-hydroxy-16α-(¹²⁵I)iodowortmannin: Identification of the Gβγ-sensitive isoform as a complex composed of 45- and 100-kda subunits

A fungal metabolite, wortmannin, is a potent inhibitor of phosphatidylinositol (PI) 3-kinases. In the present study, we prepared a radio-labeled derivative of wortmannin, 17β-hydroxy-16α-[¹²⁵I] iodowortmannin. The compound bound tightly to a 110-kDa subunit in the previously identified isoform of PI 3-kinase (p85/p110), and also to a 100-kDa peptide in a partially purified preparation of another isoform of PI 3-kinase whose activity was markedly stimulated by the βγ subunits of GTP-binding proteins (Gβγ). The binding to both peptides was inhibited by non-radiolabeled wortmannin and also by LY294002, another inhibitor of PI 3-kinases. An antibody against p85 recognized a 46-kDa peptide in the Gβγ-sensitive isozyme and precipitated the 100-kDa peptide specifically labeled with 17β-hydroxy-16α-[¹²⁵I] iodowortmannin. These results suggested that the newly found isozyme was a complex composed of 46-kDa and 100-kDa peptides.